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WO2025045826A1 - Composés pour l'activation d'une signalisation fgfr1 - Google Patents

Composés pour l'activation d'une signalisation fgfr1 Download PDF

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Publication number
WO2025045826A1
WO2025045826A1 PCT/EP2024/073830 EP2024073830W WO2025045826A1 WO 2025045826 A1 WO2025045826 A1 WO 2025045826A1 EP 2024073830 W EP2024073830 W EP 2024073830W WO 2025045826 A1 WO2025045826 A1 WO 2025045826A1
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cells
nmr
mhz
mmol
compound
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Inventor
Mikhail FEOFANOV
Juergen PAHL
Florence Andrée Suzanne HIAULT
Mohammed BENABDERRAHMANE
Andrea SALTALAMACCHIA
Michael Josef ZIEGLER
Gerrit DAUBNER
Clare Elizabeth HENRY
Karsten Koehler
Sophie Konstanze LAMBERTZ
Hamid Reza Noori
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Cultivated B GmbH
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Cultivated B GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
    • C07D213/82—Amides; Imides in position 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/16—Compounds containing any of the groups, e.g. aminoguanidine
    • C07C281/18—Compounds containing any of the groups, e.g. aminoguanidine the other nitrogen atom being further doubly-bound to a carbon atom, e.g. guanylhydrazones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/04—Systems containing only non-condensed rings with a four-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/36—Systems containing two condensed rings the rings having more than two atoms in common
    • C07C2602/38—Systems containing two condensed rings the rings having more than two atoms in common the bicyclo ring system containing five carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present invention relates to compounds of formula (I) for activating the Fibroblast Growth Factor Receptor 1 (FGFR1) signaling pathway. Moreover, it refers to the use of such compounds (I) as a culture media component for the cultivation of living cells or as a medicament for treatment and/or prevention of different diseases.
  • FGFR1 Fibroblast Growth Factor Receptor 1
  • animal and human cell cultures i.e., the cultivation of cells of animal or human origin in vitro
  • industrial productions e.g., of biopharmaceuticals such as, e.g., antibodies, hormones and other cellular products
  • stem cell techniques in in vitro fertilization, in preparation of implantable cells, and in the production of cultivated substitutes for meat and other animal-like comestible goods
  • animal and human cell cultures are of high interest.
  • animal cells including human cells
  • stimulation typically proliferate. This is typically achieved by the addition of serum, which is obtained from processed animal blood.
  • the amount of serum is typically set to a high level to reach the sufficient content of one or more stimulating growth factors.
  • the present invention is, inter alia, based on the surprising finding that compounds of formula (I) and salts thereof as defined in the present invention when added to cell culture significantly enhance proliferation of the cells. It is understood that the compounds of formula (I) are efficient activators of the FGFR1 signalling pathway leading to this proliferation. Accordingly, the compounds of formula (I) are useful for supporting the cultivation of cells. Thereby, the compounds may fully or partly substitute FGF2. Some of these compounds have already been described in the art for different applications (see below).
  • the core moiety A is selected from o an optionally substituted C5-18 aromatic group, with 1 to 3 optionally fused rings; wherein 0 to 4 C atoms are replaced by heteroatoms, wherein the heteroatoms are individually selected from N, O, and S; o an optionally substituted Ce-is arylalkyl group wherein 0 to 4 C atoms are replaced by heteroatoms, wherein the heteroatoms are individually selected from N, O, and S, an optionally substituted C3-12 cycloalkyl group, with 1 to 6 rings optionally fused, bridged or strained, wherein 0 to 4 C atoms are replaced by heteroatoms, wherein the heteroatoms are individually selected from N, O, S and Si; o an optionally substituted, straight or branched, saturated or unsaturated C2-20 aliphatic group, wherein 0 to 4 C atoms are replaced by heteroatoms, wherein the heteroatoms are individually selected from N, O, S, and Si; o an aromatic or ali
  • linkers Bi and B2 are individually selected from an ester, an ether, an amine, a thioether, an amide; and a sulfonamide;
  • tripod moieties Ci and C2 are individually selected from: o an optionally substituted C5-6 aromatic group, wherein 0 to 3 C atoms are replaced by heteroatoms, wherein the heteroatoms are individually selected from N, O, and S; o an optionally substituted C3-6 cycloalkyl group, wherein 0 to 3 C atoms are replaced by heteroatoms, wherein the heteroatoms are individually selected from N, O, and S;
  • Ci and C2 may contain one to three substituents, which are independently selected from the group consisting of hydrogen, alkyl and alkoxy,
  • a compound of formula (I) or a pharmaceutically acceptable salt thereof may be used in various medical applications, in particular for the treatment of diseases or conditions of patients, which may be treated or ameliorated by activation of the FGFR1 signaling pathway in cells of the patient.
  • the invention provides a compound of formula (I) as defined in the first aspect, or a pharmaceutically acceptable salt thereof for use in medical treatment, wherein the medical treatment is a method of wound healing, a method of organ regeneration in transplantation medicine, a method of treating a burned lesion, a disease associated with disturbed cell renewal, or a disease associated with muscle wasting, wherein the treatment or prevention may optionally comprise tissue engineering.
  • the invention provides a compound (I) as defined in the first aspect, provided that the compound is not 1-1 , I-8, 1-16, 1-17, I-75, I-76, I- 116 or 1-117.
  • the compounds of formula (I) may be used as cell culture medium supplement, such as, e.g., for cultivated meat production.
  • nutrients sufficient for cell growth including at least one carbon source and one nitrogen source,
  • (C) at least one compound of formula (I) as defined in the first aspect or a pharmaceutically acceptable salt thereof, preferably from in a concentration of 0.05 to 10 pM, preferably in a concentration of 0.1 to 5 pM, more preferably, in a concentration of 0.2 to 3 pM, and
  • the present invention also allows efficient cultivation of cells.
  • the invention provides a method for the cultivation of cells, comprising the steps of
  • step (iii) subjecting the cell culture medium of step (ii) to conditions sufficient for establishing cell growth and/or for cell differentiation;
  • the invention provides a food product comprising cultivated cells obtainable by the process according the fifth aspect or material derived therefrom, preferably wherein the food product is cultivated meat or a drinkable composition.
  • Fig. 3 Optimization of proliferative activity of 1-1 in NIH/3T3 cells using I-53 as an example. This compound showed an approx. 4.5-fold EC50 improvement over 1-1 from 0.9 to 0.2 pM.
  • Fig. 4 Dose-response with 1-1 using the Luciferase reporter assay in HEK293T cells. Upon treatment with 1-1 alone, an increase in luminescence was observed at compound concentrations higher than 3 pM. When treated with 1-1 and FGFR1 inhibitor PD166866 a signal decrease was observed. This indicates that 1-1 works partly through the FGFR1 pathway.
  • the x-axis depicts the concentration of 1-1 in pM, the y-axis shows the normalized luminescence signal (Signal/ Negative Control).
  • Fig. 5 Determination of FGFR1 dependency in NIH/ 3t3 cells.
  • PD166866 did not change the proliferative effect observed for 100 ng/ mL EGF and 2 ng/ mL TGF-pi , underlining the specificity of the observed effect.
  • the x-axis depicts the concentration of PD166866 in pM, the y-axis shows the normalized luciferase signal (signal/ negative control).
  • Fig. 7 Competition assay between recombinant FGFR1-Fc and cellular FGFR1 for FGF2 or 1-1.
  • Recombinant FGFR1-Fc inhibited the proliferative effect of (A) 1.4 pM 1-1 and (B) 100 ng/ mL FGF2, while Fc alone did not have any effect.
  • Fig. 8 Determination of 1-1 stability assessed by loss of proliferative activity and compared to FGF2.
  • 1-1 was stable over the course of 14 days in the cell culture incubator.
  • FGF2 wildtype showed decreased stability over the course of 14 days in the cell culture incubator. This reveals that 1-1 is more stable than FGF2 wildtype at 37°C/ 5% CO2.
  • Fig. 9 Measurement of cytotoxicity by LDH release and cell viability by ATP quantification upon treatment with 1-1. A change in the dead and live cell fraction was observed at 30
  • the x-axis depicts the concentration of compound in pM in logarithmic scale, the left y-axis shows the cytotoxicity calculated as: (Experimental LDH Release - Medium Background)/ (Maximum LDH Release Control - Medium Background); and the right y-axis show the viability calculated as: Treated cells/ DMSO control.
  • the moieties in the compound of the invention may optionally be substituted with one or more substituents, such as are illustrated generally herein, or as exemplified by particular classes, subclasses, and species of the invention. It will be appreciated that the phrase “optionally substituted” is used interchangeably with the phrase “substituted or unsubstituted.” In general, the term “substituted”, whether preceded by the term “optionally” or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent.
  • an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • a substituent connected by a bond drawn from the center of a ring means that the substituent can be bonded to any position in the ring.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, recovery, purification, and use for one or more of the purposes disclosed herein.
  • a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40° C. or less, in the absence of moisture or other chemically reactive conditions, for at least a week.
  • aliphatic or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched), branched, or cyclic, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation that has a single point of attachment to the rest of the molecule.
  • aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms, and in yet other embodiments aliphatic groups contain 1-4 aliphatic carbon atoms. Aliphatic groups may be linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups.
  • Aliphatic groups may also be cyclic or have a combination of linear or branched and cyclic groups. Examples of such types of aliphatic groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, CH2-cyclopropyl, CH2CH2CH(CH3)- cyclohexyl.
  • cycloaliphatic refers to a monocyclic Ca-Cs hydrocarbon or bicyclic C8-C12 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule wherein any individual ring in said bicyclic ring system has 3-7 members.
  • cycloaliphatic groups include, but are not limited to, cycloalkyl and cycloalkenyl groups. Specific examples include, but are not limited to, cyclohexyl, cyclopropenyl, and cyclobutyl.
  • heterocycle means nonaromatic, monocyclic, bicyclic, or tricyclic ring systems in which one or more ring members are an independently selected heteroatom.
  • the “heterocycle”, “heterocyclyl”, or “heterocyclic” group has three to fourteen ring members in which one or more ring members is a heteroatom independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members.
  • heterocycles include, but are not limited to, 3-1 H-benzimidazol-2-one,
  • 2-imidazolidinyl 4-imidazolidinyl, 5-imidazolidinyl, indolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothiolane, benzodithiane, and
  • Cyclic groups (e.g. cycloaliphatic and heterocycles), can be linearly fused, bridged, or spirocyclic.
  • heteroatom means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl)).
  • unsaturated means that a moiety has one or more units of unsaturation.
  • unsaturated groups can be partially unsaturated or fully unsaturated. Examples of partially unsaturated groups include, but are not limited to, butene, cyclohexene, and tetrahydropyridine.
  • Fully unsaturated groups can be aromatic, anti-aromatic, or non-aromatic. Examples of fully unsaturated groups include, but are not limited to, phenyl, cyclooctatetraene, pyridyl, thienyl, and 1-methylpyridin-2(1 H)-one.
  • alkoxy refers to an alkyl group, as previously defined, attached through an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.
  • haloalkyl refers to alkyl, alkenyl or alkoxy, as the case may be, substituted with one or more halogen atoms. This term includes perfluorinated alkyl groups, such as — CF3 and — CF2CF3.
  • halogen means F, Cl, Br, or I.
  • aryl used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members.
  • aryl may be used interchangeably with the term “aryl ring”.
  • heteroaryl used alone or as part of a larger moiety as in “heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members.
  • heteroaryl may be used interchangeably with the term “heteroaryl ring” or the term “heteroaromatic”.
  • heteroaryl rings include, but are not limited to, 2-furanyl,
  • in silico describes experiments or studies conducted through computational methods, using computer software and algorithms to analyze and predict biological, chemical, or physical processes. In silico methods are often employed to complement or guide in vitro and in vivo experiments, providing insights into complex systems or facilitating the design of new inventions. For example, computational simulations of protein interactions or drug docking studies are considered in silico experiments.
  • orthosteric refers to the primary or main active site on a receptor or enzyme where a ligand or substrate typically binds.
  • Each receptor can be activated by several FGFs. In many cases, the FGFs themselves can also activate more than one receptor (i.e., FGF1 , which binds all seven principal FGFRs (Ornitz et al. 1996). FGF7, however, can only activate FGFR2b (Duchesne et al. 2006), and FGF18 was recently shown to activate FGFR3 (Davidson et al. 2005).
  • FGF2 (or b-FGF) is an 18 kD protein and is one of the most studied natural modulators of FGFR1 activity. FGF2 mediates a broad spectrum of mitogenic and pro-survival effects (Nawrocka et al. 2020) and, thus, promotes different cellular phenotypes (e.g. survival, proliferation, migration, invasion, angiogenesis, sternness, maturation) in a multitude of cell types (e.g. stem cells, fibroblasts, skeletal muscle cells, endothelial cells) in a context-dependent manner. FGF2 binding to FGFR1 may lead to conformational changes of FGFR1 that allow receptor dimerization.
  • b-FGF is an 18 kD protein and is one of the most studied natural modulators of FGFR1 activity. FGF2 mediates a broad spectrum of mitogenic and pro-survival effects (Nawrocka et al. 2020) and, thus, promotes different cellular phenotypes (e.g
  • the compounds of the general formula (I) are composed of core moiety A, which is connected by two linkers Bi and B2 to the two tripod moieties Ci and C2. Each tripod moiety is connected to two active moieties Di and D2 or D3 and D4 respectively.
  • the compound of formula 1-1 was discovered via structure-based virtual screening using a proprietary algorithm as a molecule that potentially binds to and activates FGFR1. Based on the in silico study, it is inferred that the guanidines are among the key chemical groups responsible for activity. This is experimentally confirmed in Example 2 showing that a further compound of the formula (I), namely compound 1-17, which has a very different core structure as compared to compound 1-1 , has a comparable effect.
  • the active moieties Di, D2, D3 and D4 are H or wherein Di and D2 may not both be hydrogen and D3 and D4 may not both be hydrogen. According to a preferred embodiment, all four the active moieties Di , D2, D3 and D4 are
  • the core moiety A is optionally substituted Cs-is aryl, i.e. C 5 , Cg, C7, Cg , C9 , C 10 , C11, C12 , C13 , C14, C15, Ci6, C17, or Cig . with 1 , 2 or 3 optionally fused rings.
  • the rings may be fused or bound.
  • 0 to 4 C atoms i.e. 0, 1 , 2, 3, or 4 C atoms, may replac-ed by heteroatoms, wherein the heteroatoms are individually selected from N, O, and S.
  • the core moiety A is an optionally substituted Cs-saryl wherein 0 to 3 C atoms are replaced by heteroatoms, wherein the heteroatoms are individually selected from N, O, and S.
  • the core moiety A is an optionally substituted six-membered aryl or heteroaryl.
  • the Cs heteroaryl may have one or two heteroatoms.
  • the heteroatom is N.
  • the B groups are in meta position of the six-membered aryl or heteroaryl. Accordingly, the B groups are separated by one carbon atom on the six-membered aryl or heteroaryl. This corresponds to the 1 ,3-positions.
  • the B groups are in para position of the six-membered aryl or heteroaryl.
  • A is a benzene ring.
  • the core moiety A is an optionally substituted Ce-is, arylalkyl group, i.e. a Cg, C7, C 8 , C 9 , C10 , On, Ci 2 , C13 , C14, C15, Ci6, C17, or Cig, aralkyl.
  • arylalkyl group 0 to 4 C atoms, i.e. 0, 1 , 2, 3, or 4 C atoms, may be replaced by heteroatoms.
  • the heteroatoms are individually selected from N, O, and S.
  • the aralkyl is a six-membered aryl or heteroaryl ring with two C1-3 alkyl groups attached.
  • the alkyl groups may be methyl, ethyl or propyl, preferably methyl.
  • A contains two benzene rings.
  • A is selected from a dibenzyne, biphenyl, diphenylmethane or diphenyl ether.
  • the core moiety A is an optionally substituted C3-12 cycloalkyl group i.e. a C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , CH, or C 12 , with 1 to 6 rings, i.e. 1 , 2, 3, 4, 5, or 6 rings.
  • the rings may optionally be fused, bridged or strained.
  • 0 to 4 C atoms i.e. 0, 1 , 2, 3, or 4 C atoms, may be replaced by heteroatoms,
  • the heteroatoms may be individually selected from N, O, S and Si.
  • the core moiety A is an optionally substituted C3-6 cycloalkyl, i.e. an optionally substituted cyclopropane, cyclobutane, cyclohexane or cyclopentane.
  • the C3-6 cycloalkyl may be bridged.
  • 0 to 1 C atoms may be replaced by a heteroatom.
  • the heteroatoms are individually selected from N, O, and S.
  • the C3-6 cycloalkyl does not contain heteroatoms.
  • the core moiety A is an optionally substituted, straight or branched, saturated or unsaturated C2-20 aliphatic group, i.e. C 2 , C3, C 4 , C5, C 6 , C 7 , C 8 , C 9 , C10 , C11, C12 , C13 , C14, Cis, Ci6, C17, Cig, Ci9, or C20.
  • C2-20 aliphatic group i.e. C 2 , C3, C 4 , C5, C 6 , C 7 , C 8 , C 9 , C10 , C11, C12 , C13 , C14, Cis, Ci6, C17, Cig, Ci9, or C20.
  • 0 to 4 C atoms i.e. 0, 1 , 2, 3, or 4 C atoms, are replaced by heteroatoms.
  • the heteroatoms are individually selected from N, O, S, and Si.
  • the C2-20 aliphatic group is saturated.
  • the core moiety A is an optionally substituted, straight or branched, saturated or unsaturated C2-12 aliphatic group, wherein 0 to 4 C atoms are replaced by heteroatoms, wherein the heteroatoms are individually selected from N, O, and S.
  • the C2-12 aliphatic group is saturated.
  • the C2-12 aliphatic group is straight.
  • a substituent is in ortho position to Bi or B 2 .
  • a substituent is in ortho position to Bi and B 2 .
  • first substituent is in meta position to Bi and B 2 .
  • the core moiety A is an aromatic or aliphatic crown ether.
  • the core moiety A is an aromatic crown ether.
  • the crown ether may be a 18-crown-6 ether.
  • the core moiety A is optionally substituted, which means that the core moiety A may contain one to three substituents, which are independently selected from the group consisting of halogen, amide, amine, nitro, cyano, hydroxyl or hydrocarbyloxy, or aldehyde, ketone, cycloalkyl, carboxyl, ether, ester, optionally halogenated alkyl, alkenyl, alkenylaryl, alkenyl aryl, alkynyl, alkoxy, alkylthio, sulfonyl, sulfonylamide.
  • A may contain one to three substituents, which are independently selected from the group consisting of halogen, hydrogen, hydroxyl, methyl, methoxy, optionally halogenated Ci- 6 alkyl, optionally halogenated Ci- 6 alkoxy, C5-6 cycloalkyl, in which 0 to 2 C atoms are replaced by heteroatoms and amine.
  • the substituent is a polyether.
  • the substituent is selected from: wherein X is N, O, S, preferably O.
  • A contains three halogen substituents.
  • the halogens are F.
  • the Ci- 6 alkyl is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, neohexyl.
  • the substituent is a tert-butyl, preferably in meta position to both Bi and B2.
  • the Ci- 6 alkyl is halogenated by one to three of fluorine, chlorine, bromine or iodine.
  • the halogenated alkyl is trifluoromethyl, preferably in meta position to both Bi and B2.
  • the halogenated alkyl is difluoromethyl, preferably in meta position to both Bi and B2.
  • the halogenated alkoxy is trifluoromethoxy, preferably in meta position to both Bi and B2.
  • the substituent is morpholine, preferably in ortho or meta postion to Bi.
  • the alkylthio group is methylthio, preferably in meta position to both Bi and B2.
  • the sulfonyl group is methylsulfonyl, preferably in meta position to both Bi and B2.
  • the substituent is styryl, preferably in ortho or meta position to Bi.
  • the cycloal kylethynyl is selected from cyclohexylethynyl or a cyclopropylethynyl and piperidineethynyl, preferably in meta position to both Bi and B2.
  • the alkynyl is methylacetylene, preferably in meta position to both Bi and B2.
  • the core moiety A is selected from the following residues (the dashed lines indicate the bonds to Bi and B2):
  • X may be N, O, or S.
  • X is N.
  • Rs may be O, S, or NRTM, with R w being H or C1-6 alkyl.
  • Ra is NRTM.
  • R3 is H.
  • R2 may be H, or C1-6 alkyl.
  • the amine NR5R6 may be selected from:-
  • R? may be halogen, methyl, methoxy, tert-butyl, NO2, CN, CF3, OCF3, CF2, COOH, COOCH3, SCH 3 , CO2NH2, N(CH 3 ) 2 , NH 2 , SO2CH3, NHCOOCH3, NHSO 2 CH3, and NHSO2CF3, and CONR11R12 with R11 and R12 being independently selected from H and C1-6 alkyl.
  • Rn and R12 are both H.
  • Rn and/or R12 are methyl.
  • Rs may be H or phenyl. Preferably, Rs is H.
  • R9 may be methyl, Cs-Cs cycloalkyl, piperidinyl or hydroxyisopropyl.
  • Rg is methyl.
  • Xi may be oxygen or methylene.
  • Xi is oxygen.
  • A is not a polyether. According to one embodiment, A is not selected from: wherein X is N, O, S, preferably O.
  • At least one of Ci and C2 and preferably both of Ci and C2 are both pyridinyl groups.
  • the compound of formula (I) is selected from 1-1 , I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, 1-10, 1-11 , 1-15, 1-16, 1-17, 1-18, 1-19, I-20, I-22, I-23, I-24, I-25, I-26, I-27, I-28, I-29, I-30, 1-31 , I-32, I-33, I-34, I-35, I-36, I-37, I-38, I-39, I-40, 1-41 , I-42, I-43, I-44, I-45, 1-46, 1-47, 1-48, 1-49, 1-50, 1-51 , I-52, I-53, I-54, I-55, I-56, I-57, I-58, I-59, I-60, 1-61 , I-62, I-63, I-64, I-65, I-66, I-67, I-68, I-69, I-70, 1-71
  • a pharmaceutically acceptable salt may be understood in the broadest sense as any salt of the respective compound of formula (I) that is reasonably usable in a pharmaceutical context.
  • pharmaceutically acceptable does not necessarily mean that the respective component is indeed used in a pharmaceutical context. This rather refers to the suitability to do so.
  • a pharmaceutically acceptable salt or other component or composition will typically also be inherently usable as being cosmetically acceptable.
  • a pharmaceutically acceptable salt or other component or composition typically bears a low toxicity and can be administered to a human or non-human animal (typically mammal or avian) body without seriously harming this human or non-human animal as well as to a cell culture without seriously harming this cell culture, when administered in reasonable concentrations as used for the compound of formula (I).
  • a concentration higher than 10 pM could lead to wastage of expensive reagents without necessarily improving cell culture outcomes.
  • a concentration below 0.05 pM might not stimulate enough cell growth and proliferation.
  • the compound of formula (I) is present in a concentration of 0.1 to 5 pM. Maintaining concentrations within a defined range can be cost-effective as it ensures that the appropriate amount of growth factor is used - not too much (wasteful) or too little (ineffective)
  • the concentration of the compound of formula (I) is 0.2 to 3 pM. A concentration in this range ensures that cells have sufficient quantities of the growth factor to promote cell proliferation and growth.
  • the actual concentration of the compound of formula (I) can depend on various factors, including the intrinsic factors such as cell type, genetic factors, age and passage number, and health status and extrinsic factors such as nutrient availability, oxygen levels, temperature, CO2 concentration, culture vessel and surface cell density. Therefore, the most suitable concentration can be chosen based on these considerations to optimize the therapeutic effects of the compound.
  • the compounds of the invention may be prepared as described in Example 1. Furthermore, compounds 1-1 , I-8, 1-16 1-17, 1-117 and their methods of synthesis have been described in US 5,599,984 A, which is incorporated herein by reference. Compounds I-75, I-76 and 1-116 as well as their methods of synthesis have been described in Schroder et al. 2016, which is incorporated herein by reference. Compound 1-117 and its method of synthesis has been described in US 5,750,573, which is incorporated herein by reference.
  • the compounds of formula (I) may be used for treatment of diseases or conditions of patients, which may be treated or ameliorated by activation of the FGFR1 signaling pathway in cells of the patient. This activation leads to an increase in cell proliferation in the patient.
  • the compounds may in particular be useful for the treatment of the following disorders of conditions:
  • Osteoporosis is characterized by a reduction in bone density due to an imbalance in the bone remodeling process. Compounds that promote the proliferation of osteoblasts (bone-forming cells) can potentially restore this balance, leading to increased bone formation and improved bone density. In osteoporotic patients, fractures are a significant concern. Enhancing cell proliferation might also accelerate the fracture healing process. FGFR1 signaling has a role in bone formation and maintenance. Increasing its signaling could potentially promote osteoblast activity, which may be beneficial in conditions of decreased bone density or osteoporosis.
  • FGFR1 signaling has been implicated in the survival and regeneration of certain neuron populations. Hence, enhancing its activity could be explored in the context of promoting neural regeneration or protecting neurons in diseases like Parkinson's. In conditions like Parkinson's disease, Alzheimer's disease, or Huntington's disease, where specific neuronal populations are lost, stimulating the proliferation of neural progenitor cells or specific subsets of neurons might have therapeutic benefits. In the case of nerve injuries or certain degenerative conditions like peripheral neuropathies, compounds that promote the proliferation of neurons or supporting cells (like Schwann cells) can aid in neural tissue regeneration.
  • the compounds of formula (I) that increase FGFR1 signaling will have a role in enhancing organ regeneration or repair in transplantation medicine.
  • FGFR1 signaling can promote angiogenesis, which is the formation of new blood vessels. Compounds that enhance this signaling pathway could potentially speed up the process of revascularization in the transplanted organ. Stimulating cell proliferation and differentiation: Proper organ function often requires the growth and differentiation of specific cell types. FGFR1 signaling can support the proliferation of progenitor cells and guide the differentiation of certain cell lineages, aiding in tissue repair and regeneration. Reduction of fibrosis: FGFR signaling might help reduce tissue fibrosis, a process where normal tissue is replaced with scar tissue.
  • fibrosis In the context of organ transplantation, controlling fibrosis can be crucial for the long-term success of the transplant, as excessive fibrosis can impair organ function. Protection against ischemia-reperfusion injury: This type of injury can occur when blood supply returns to the tissue after a period of lack of oxygen (ischemia). It's a significant concern in organ transplantation. FGFR signaling has the potential to protect tissues against such injuries.
  • Pre-transplant Organ Cultivation For organs grown ex vivo (outside the body), such as in bioengineered organs or tissues, promoting cell proliferation can accelerate the growth and maturation of the organ, making it suitable for transplantation sooner.
  • Posttransplant Organ Integration After an organ is transplanted, it is crucial that it integrates well with the recipient's body. Compounds that promote cell proliferation could aid in the faster establishment and growth of the transplanted organ, improving its function and longevity.
  • Enhancing FGFR1 signaling will have therapeutic effects in the contexts of burned lesions: Wound Healing: FGFR signaling plays roles in promoting tissue repair and wound healing. Upregulating FGFR1 signaling could potentially accelerate the healing process in burn wounds. Promoting cell proliferation can accelerate the healing process of the burn wound by enhancing tissue regeneration. Angiogenesis: Restoration of blood flow to burned tissue is essential for healing. FGFR1 can promote angiogenesis (formation of new blood vessels), which may speed up revascularization of the affected tissue. Reduction of Scarring: FGFR signaling might help modulate the wound healing response in a manner that reduces excessive fibrosis and scarring, which are common complications of severe burns.
  • Promoting cell proliferation can by facilitating a more efficient regenerative process, the formation of scar tissue might be minimized.
  • Enhancing FGFR1 signaling will have a therapeutic effect in the contexts of diseases with disturbed cell renewal: Skin disorders: For conditions like psoriasis, where there is disturbed skin cell renewal, modulating FGFR1 signaling could help normalize the skin cell growth cycle. Moreover, conditions like psoriasis or eczema can benefit from compounds that promote skin cell proliferation, leading to the renewal and repair of skin layers.
  • Hair Growth FGFR signaling is involved in hair follicle regulation. If disturbances in hair growth are related to compromised FGFR signaling, enhancing this pathway might be beneficial. Certain hair loss disorders result from a disturbance in hair follicle cell proliferation. Promoting cell growth can potentially stimulate hair regrowth.
  • Enhancing FGFR1 signaling will have a therapeutic effect in the contexts of Diseases associated with muscle wasting.
  • Promotion of Muscle Cell Growth FGFR1 signaling has roles in muscle cell differentiation and proliferation. In diseases such as muscular dystrophy or in conditions like cachexia (seen in some cancer patients), increasing FGFR1 signaling could potentially promote muscle regeneration. Promoting muscle cell proliferation can potentially counteract muscle atrophy seen in diseases like muscular dystrophy or conditions like cachexia. Stimulation of satellite cells: Satellite cells are muscle stem cells that play a crucial role in muscle repair. Compounds that promote their proliferation can be beneficial for muscle recovery. FGFR1 signaling might help in the activation and differentiation of these cells, aiding muscle repair and regeneration.
  • the invention provides a compound of formula (I) as defined in the first aspect, or a pharmaceutically acceptable salt thereof for use in medical treatment, wherein the medical treatment is a method of wound healing, a method of organ regeneration in transplantation medicine, a method of treating a burned lesion, a disease associated with disturbed cell renewal, or a disease associated with muscle wasting, wherein the treatment or prevention may optionally comprise tissue engineering.
  • the second aspect relates to a method of treatment wherein the medical treatment is a method of wound healing, a method of treating osteoporosis, a method of organ regeneration in transplantation medicine, a method of treating a burned lesion, a disease associated with disturbed cell renewal, or a disease associated with muscle wasting, wherein the treatment or prevention may optionally comprise tissue engineering.
  • a medicament of the present invention may have any galenic form.
  • a medicament may be in any dosage for e.g., an ingestible composition (e.g., a pill, a dragee, a syrup, a drinkable liquid), an inhalable composition (e.g., a spray), a dosage form penetrating the skin (e.g., a cream, a lotion, a plaster, a suppository, an eye drop, a spray), or an injectable composition. It is preferably (essentially) sterile and is preferably a-pyrogenic.
  • the patient suffers from a disease associated with (dysregulated) cellular growth and/or FGF-mediated (in particular FGF2-mediated) activity, and/or disturbed FGFR signaling (in particular FGFR1 signaling).
  • a disease associated with (dysregulated) cellular growth and/or FGF-mediated (in particular FGF2-mediated) activity and/or disturbed FGFR signaling (in particular FGFR1 signaling).
  • the invention provides a compound (I) as defined in the first aspect, provided that the compound is not 1-1 , I-8.I-9 1-16,1-17, I-75, I-76, 1-116 or 1-117.
  • the core moiety A is not a Ge aryl or C2-8 unsubstituted alkane. According to one embodiment, Ci and C2 are not both benzyl rings. According to one embodiment, the core moiety A is not a six-membered aryl or heteroaryl or a C2-8 unsubstituted alkane.
  • the cell culture medium The cell culture medium
  • the compounds of formula (I) may be used in specialized cell culture media for survival, proliferation, migration, invasion, angiogenesis, sternness, and differentiation of animal cells.
  • a cell culture may benefit from the use of one or more compounds of formula (I) or pharmaceutically acceptable salt thereof.
  • Cells of interest may be contacted with the one or more compounds of formula (I) or pharmaceutically acceptable salts thereof by any means.
  • the cells of interest may be contacted with the one or more compounds of formula (I) or pharmaceutically acceptable salts thereof by the presence of the latter in a cell culture medium.
  • Such cell culture medium bears technically special characteristics.
  • the present invention relates further to a cell culture medium comprising at least one compound of formula (I) or a pharmaceutically acceptable salt thereof as defined in the first aspect.
  • the invention provides a cell culture medium comprising at least one compound of formula (I) or a pharmaceutically acceptable salt thereof as defined in the first aspect, preferably wherein the cell culture medium comprises or consists of:
  • nutrients sufficient for cell growth including at least one carbon source and one nitrogen source,
  • (C) at least one compound of formula (I) as defined in the first aspect or a pharmaceutically acceptable salt thereof, preferably from in a concentration of 0.05 to 10 pM, preferably in a concentration of 0.1 to 5 pM, more preferably, in a concentration of 0.2 to 3 pM, and
  • a cell culture medium may be any medium that is suitable to allow maintenance and viability and preferably proliferation of cells of interest.
  • the person skilled in the art knows a variety of commercially available cell culture media.
  • a cell culture medium at least comprises water and nutrients.
  • the cell culture medium of the present invention may comprise any contents of at least one compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • it may comprise 0.01 nM to 10 mM, or 0.05 nM to 1 mM, or 0.1 nM to 100 pM, or 0.5 nM to 50 pM, or 1 nM to 10 pM, or 5 nM to 1 pM, or 10 nM to 500 pM, of at least one compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • a cell culture medium typically has an osmolarity that is near by the isotonic osmolarity to avoid disturbance of cells.
  • a cell culture medium typically has a pH range that is near by the neutral range and slightly basic such as, e.g., in the range of pH 6.0 to 8.0, or pH 6.5 to 7.8, or pH 7.0 to 7.7, or pH 7.2 to 7.5, or (approximately) pH 7.4.
  • the person skilled in the art knows adequate buffers to achieve such pH range such a hydrogen phosphate/phosphate buffers, MES (2-(N-morpholino)ethanesulfonic acid) buffers, etc.
  • the buffer system may also comprise an open buffer based on, e.g., addition of gaseous CO2 (e.g., approximately 5% during cultivation of the cells).
  • the person skilled in the art will be able to choose and use commercially available cell culture media accordingly.
  • the compounds of formula (I) may be used as cell culture medium supplement such as, e.g., for cultivated meat production.
  • the compounds may particularly support serum-free cell culture media and may partly replace growth factor activities, in particular FGF activities and thereby of serum.
  • the cell culture medium may be liquid, viscous, or solid.
  • a cell culture medium is a liquid cell culture medium.
  • it may be a (hydro)gel on or in which cells are cultivated, either in contact with air or covered by another cell culture medium, which is preferably liquid.
  • one or more compounds of formula (I) or a pharmaceutically acceptable salts thereof may partly or completely substitute one or more growth factors such as FGF, in particular FGF2. Therefore, culturing cells when omitting such one or more growth factors may be enabled.
  • the cell culture medium is further characterized in that it does (essentially) not contain serum of animal origin, any subtype of FGF of animal origin, any peptide growth factor of animal origin, in particular any peptide growth factor or steroid growth factor of animal origin, in particular any steroid growth factor.
  • the composition of serum is complex and not fully defined. It contains various growth factors, hormones, and other components that can influence cell behavior, but their exact concentrations and roles may not be well-understood. This lack of definition can lead to difficulties in interpreting experimental results. Serum composition can vary significantly between different batches and sources, leading to inconsistencies in cell culture conditions. This variability can affect cell growth, differentiation, and experimental outcomes, making it challenging to reproduce results. Proteins derived from animal sources, such as bovine serum albumin (BSA) or bovine transferrin, can also exhibit batch-to- batch variability. Similar to serum, proteins from animal origin can be expensive, especially if large quantities are required for extensive cell culture studies.
  • BSA bovine serum albumin
  • the cell culture medium does (essentially) not contain FGF2, in particular no FGF2 of animal origin.
  • FGF2 is the compound to be replaced by the compounds of the invention.
  • the combination of FGF2 and the compounds of the invention does not have an additive effect.
  • the present invention also allows efficient cultivation of cells.
  • the invention provides a method for the cultivation of cells, comprising the steps of
  • step (iii) subjecting the cell culture medium of step (ii) to conditions sufficient for establishing cell growth and/or for cell differentiation;
  • the cells preferably comprise FGFR1 , which signaling pathway is activated by a compound of formula (I) as defined in the first aspect.
  • the cells may be from human or animal source.
  • the cells may be for example obtained from the following animals: cattle (bovine), the cells of which are one of the primary sources for cultivated beef production; Chicken (poultry), the cells of which are used to produce cultivated chicken meat; pigs (swine), the cells of which are used for cultivated pork production; fish (e.g., salmon, tuna), the cells of can be used to produce cultivated fish fillets; ducks, the cells of which can be a potential source for cultivated duck meat; sheep, the cells of which can be used to produce cultivated lamb or mutton; rabbits, the cells of which can be used to produce cultivated rabbit meat; quails: quails are a potential source for cultivated quail meat; turkey, the cells of which can be used to produce cultivated turkey meat; deer, the cells of which cells can be used to produce cultivated venison.
  • Myoblasts are muscle precursor cells that can differentiate and fuse to form multinucleated myotubes, which eventually mature into muscle fibers. Myoblasts are a primary choice for cultivated meat production due to their natural ability to develop into muscle tissue. Satellite cells are a type of stem cell found in skeletal muscle. These cells play a crucial role in muscle regeneration and repair. When activated, satellite cells can proliferate and differentiate into myoblasts, making them suitable candidates for cultivated meat.
  • Addition of the compound of formula (I) or a pharmaceutically acceptable salt thereof may be performed by any means.
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof may be added during cultivation. It may be added as such, i.e., as pure substance, may be added in a dilution in a stock solution of higher concentration, or may be added by replacing the cell culture medium. Cultivating with the compound of formula (I) or a pharmaceutically acceptable salt thereof may be conducted once, twice or more often or may be constantly.
  • the concentrations of compound of formula (I) or a pharmaceutically acceptable salt thereof may be maintained constant during cultivation of cells or may be varied by any profile, e.g., increased or decreased over time.
  • Separating the cultivated cells from the cell culture medium may be conducted by any means. If the cells are adherently grown, these may, for instance, be detached by mechanical scraping and/or enzymatically (e.g., by using a trypsin or other digestive enzyme composition). Suspended cells may be separated from the cell culture medium by centrifugation and/or filtration (e.g., dead end filtration or cross-flow filtration).
  • the method can be included in a method of producing a food product, in particular cultivated meat.
  • the method of cell expansion further contains a step of separating the cultivated cells from the cell culture medium.
  • the method of producing cultivated meat may contain the following steps:
  • the first step in cell cultivation is to obtain cells from a living donor animal. This can be done through a biopsy or tissue sample, which typically contains muscle cells, also known as myocytes.
  • the donor animal's welfare is a crucial consideration, and efforts are made to minimize any harm during the cell collection process.
  • the next step in the generation of cultivated meat is to develop the cultured cells into functional muscle tissue.
  • the cultured cells need to differentiate into specific cell types. This process involves exposing the cells to specific biochemical and mechanical cues that mimic the natural environment of muscle tissue development.
  • a scaffold made of biocompatible materials can be used to provide structural support for the growing cells. Additionally, 3D bioprinting techniques can be employed to create complex tissue structures, enhancing the organization and functionality of the cultured meat.
  • the cultured cells are further cultivated and matured to promote the formation of muscle tissue.
  • This maturation process aims to develop a product that closely resembles traditional meat in terms of texture, taste, and nutritional content.
  • the cultivated meat reaches the desired level of maturity, it is harvested and processed into various meat products, such as burgers, sausages, or nuggets, using conventional food processing techniques.
  • the cultivated meat products are packaged and prepared for distribution to consumers or food outlets.
  • the invention provides a food product comprising cultivated cells obtainable by the process according the fifth aspect or material derived therefrom, preferably wherein the food product is cultivated meat or a drinkable composition.
  • the cultivated meat according to the invention may be for example cultivated beef, cultivated chicken, cultivated pork, cultivated fish (e.g., salmon, tuna), cultivated duck, cultivated lamb or mutton, cultivated rabbit, cultivated quail, cultivated turkey, cultivated venison.
  • cultivated beef cultivated chicken
  • cultivated pork cultivated fish
  • cultivated duck cultivated lamb or mutton
  • cultivated rabbit cultivated quail
  • cultivated turkey cultivated venison.
  • the food product is cultivated meat or a drinkable composition.
  • the cell culture medium and the food product of the present invention may be provided in any package. Depending on the intended use, it may be provided in different packaging. It may be stored at any condition suitable for this purpose such as, e.g., at ambient temperature (e.g., 18 to 30°C, preferably 18 to 25°C), in a fridge (e.g., at 0 to 15°C, preferably 3 to 10°C), in a freezer (e.g., -30 to 0°C, preferably -25 to -10°C), in a deep freezer (e.g., -100 to -300°C, preferably -90 to -55°C), on liquid nitrogen, on dry ice, or even one or more liquid noble gases. For instance, it may be provided in a flask, a bottle, another container, or a package for solid material. It may be stored at dry state, as a (hydro)gel, as a suspension, emul
  • reaction apparatuses were dried under dynamic vacuum using a heat gun, and anhydrous solvents (Sure-SealTM products from Merck KGaAr AcrosealTM products from Thermo Fischer Scientific inc.) were employed. Commercial solvents and reagents were used without further purification.
  • reaction conditions reaction time and temperature may vary.
  • TLC thin-layer chromatography
  • UHPLC-MS ultra-high performance liquid chromatography-mass spectrometry
  • the column eluate was analysed using a 1260 Infinity II Diode Array Detector WR scanning from 200 to 400 nm and LC/MSD iQ mass spectrometer scanning in both positive and negative ion modes from 140 to 1000 Da. Purifications were performed by automated flash chromatography (FC) using Pure C-810 Buchi instrument and pre-packed BGB Scorpius C18-HP 100 A or Silica 60 A cartridges or HPLC Agilent Infinity II Preparative HPLC using InfinityLab ZORBAX Eclipse Plus C18 column. UV detection was used to trigger fraction collection.
  • FC automated flash chromatography
  • Purifications may vary in general, solvents and the solvent ratios used for eluents/gradients were chosen to provide appropriate Rfs or retention times.
  • Mass spectrometry data are reported from UHPLC-MS analyses. Mass spectrometry (MS) was performed via electrospray ionization (ESI).
  • concentration refers to the removal of solvent at reduced pressure on a rotary evaporator with a bath temperature less than 60° C.
  • the abbreviations “min” and “h” stand for “minutes” and “hours,” respectively.
  • the term “TLC” refers to thin-layer chromatography, “room temperature or ambient temperature” means a temperature between 18 to 25° C., “, “UHPLC” refers to ultra-high performance liquid chromatography, “HPLC” refers to high-performance liquid chromatography, FC refers to automated flash chromatography.
  • ACN acetonitrile
  • DIPEA N,N-Diisopropylethylamine.
  • Dppf 1 , 1 -ferrocenediyl-bis(diphenylphosphine).
  • PCya tricyclohexylphosphine
  • PyBOP benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate.
  • Rpm revolution per minute.
  • TCFH Chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate
  • Methyl ester was dissolved in mixture of THF/MeOH/Water (1 :1 :1) and LiOH was added. The resulting mixture was stirred overnight. After the completion of the reaction, 1 M HCI was added (5-10 ml) and the mixture was extracted with EtOAc. The organic layers were combined, dried over sodium sulfate and evaporated under the reduced pressure obtaining intermediate.
  • Step 1
  • Step 1
  • Step 1
  • Step 1
  • Step 1
  • f-BuBrettPhos Pd G3 43 mg, 0.05 mmol
  • f-BuBrettPhos 24 mg, 0.05 mmol
  • K4[Fe(CN)e]*3H2O 184 mg, 0.5 mmol
  • 5-bromoisophthalic acid dimethyl ester 273 mg, 1 mmol
  • the vessel was evacuated and refilled with nitrogen (three cycles).
  • Degassed dioxane (2.5 mL), KOAc (12 mg, 0.125 mmol) and water (2.5 mL) were then added to the reaction tube via syringe.
  • test tube was placed in an oil bath preheated to 100 °C. After 4 h of stirring at 100 °C, the reaction mixture was then cooled to room temperature. The contents of the test tube were transferred to a separation funnel using EtOAc (15 mL) and brine (15 mL), and the organic layer was separated from the aqueous layer. The solvents were evaporated and the residue was purified by chromatography on silica gel eluting with cyclohexane to cyclohexane:ethylacetate 50:50 to obtain the product in 72% yield (158 mg).
  • Step 1
  • Step 1
  • Step 1
  • Step 1
  • Trimethyl 1 ,3,5-benzenetricarboxylate (2.00 g, 7.93 mmol) was suspended in methanol (180 mL) and I M sodium hydroxide solution (7.14 mL, 280 mg of sodium hydroxide) was added. The mixture was stirred at room temperature for 18 h. The resulting solution was concentrated under reduced pressure to afford a white solid which was partitioned between dichloromethane (150 mL) and saturated aqueous sodium, bicarbonate solution (150 mL). The organic phase was separated and was extracted with saturated aqueous sodium bicarbonate solution (150 mL) before being discarded.
  • Step 1
  • Step 1
  • Step 1 Method B.
  • Step 1
  • Step l Method B. Starting materials: 5-bromoisophthalic acid dimethyl ester (400 mg, 1.46 mmol), 6,6-difluoro-3-azabicyclo[3.1.0]hexane hydrochloride (227 mg, 1.46 mmol), CS2CO3 (0.716 mg, 2.20 mmol), 15 ml of toluene, Pd(OAc)2 (16.6 mg, 0.074 mmol), BINAP (45 mg, 0.074 mmol). MS (M+H) + : 312.1.
  • Step 1
  • Step 1
  • Step 1
  • Step 2 Synthesized according to general procedure 1. Starting materials: THF/MeOH/Water (1 ml/1 ml/1 ml) and LiOH (400 mg).
  • Step 1
  • Step 1
  • Step 1
  • Step 1
  • Step 1
  • Step 1 MS (M+H) + : 300.9.
  • Step 1
  • Step 1
  • Step 1 anhydride (12.5 mL) was cooled to -40 °C prior to the dropwise addition of 69% nitric acid (0.82 mL). The reaction mixture was warmed to room temperature and stirred for 1.5 h. The solution was then poured onto ice water and neutralized with sat. sodium bicarbonate solution. The aqueous layer was then separated and extracted with ethyl acetate. The combined organic extracts were then dried using MgSCU, filtered, then concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with cyclohexane to cyclohexane: ethylacetate 50:50 to obtain the desired product as a yellow solid in 23% yield (320 mg). MS (M+H) + : 154.8.
  • tin(ll)chloride (1.05 g, 5 mmol) was dissolved in 3 of mL cone. HCI and warmed up to 60 °C followed by the slow addition of product from the previous step (250 mg). Afterwards, the solution was stirred for further 15 min and then poured, under gas formation, in a mixture of 5 g potassium carbonate and 20 mL ice/water. The product was extracted with EtOAc and the combined organic layers were dried over sodium sulfate. Finally, the solvent was removed in vacuo obtaining the product as black solid, which was immediately used in the next step.
  • Step 1
  • Step 1
  • Step 1 Additional step between Step 1 and 2 was added if Boc-deprotection was necessary.
  • step 1 The product from step 1 was dissolved in mixture of DCM:TFA (1 ml/1ml) and stirred for 3 h at rt. After the completion of the reaction the solvents were evaporated, and the residue was used in the next step.
  • Step 1-2
  • the compounds was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compounds were synthesized according to general procedure 2 using the product from the previous step. 1-67.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the intermediate 53 (58 mg, 0.14 mmol) and diamine (0.14 mmol) were dissolved in 1 ml of MeCN. N-methyl imidazole (0.06 ml) and TCFH (73 mg, 0.28 mmol) were added sequentially. After the completion of the reaction, the solids were filtrated, washed with ethanol and used in the next step without additional purification.
  • the compounds were synthesized according to general procedure 2 using the product from the previous step. 1-121.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • the compounds were synthesized according to general procedure 2 using the product from the previous step.
  • Step 1
  • Step 1
  • Step 1
  • the compound was synthesized according to general procedure 2 using the product from the previous step.
  • Step 1

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Abstract

La présente invention concerne un composé de formule (I) et leur utilisation pour cultiver des cellules in vitro, le composé ayant la formule générale (I). En outre, l'invention concerne le composé de formule (I) destiné à être utilisé dans un traitement médical, le traitement médical étant un procédé de cicatrisation de plaie, un procédé de régénération d'organe dans la médecine de transplantation, un procédé de traitement d'une lésion brûlée, une maladie associée à un renouvellement cellulaire perturbé, ou une maladie associée à l'atrophie musculaire, le traitement ou la prévention pouvant éventuellement comprendre une ingénierie tissulaire. Les composés de formule (I) peuvent être utilisés en tant que complément de milieu de culture cellulaire. Ainsi, l'invention concerne également un milieu de culture cellulaire comprenant au moins un composé de formule (I) un procédé de culture de cellules et un produit alimentaire comprenant des cellules cultivées.
PCT/EP2024/073830 2023-08-25 2024-08-26 Composés pour l'activation d'une signalisation fgfr1 Pending WO2025045826A1 (fr)

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